Abstract: SR proteins are one of the important classes of RNA binding splicing factors involved in both constitutive and regulated splicing in mammalian cells. Our lab has been pursuing the mechanism and regulation of SR proteins. In the current award period, we have made significant conceptual advances in this research front by demonstrating a critical role of SR proteins in cell growth control and protection of genome stability, elucidating the mechanism for SR protein-mediated exon inclusion and skipping, and revealing collaboration between SR proteins and other types of splicing regulators in establishing cell type- and tissue-specific splicing programs. One of the most surprising discoveries is on an active role of SR proteins (particularly SC35) in transcriptional elongation. Our lab has also been making a systematic effort in generating conditional knockout mice to study splicing programs in development, which is essential to understand the regulation of alternative splicing under physiological settings. Based on these critical advances, we propose to continue this fruitful investigation by focusing on the following three specific aims: (1) We will determine rules that govern SR protein-dependent splice site selection in vivo. This line of investigation is important because we recently found that SR proteins are equally involved in regulated exon inclusion and skipping events in vivo, which is contrary to the common assumption that SR proteins promote exon inclusion. We will use the latest genomics approaches to determine the SR-RNA interaction network to test the hypothesis that the functional outcome may depend on the overall SR occupancy on a regulated exon relative to flanking exons. (2) We propose to define the molecular basis for determining exon identity in the genome, which has been one of the major unsolved puzzles in the field. Based on our recent genome-wide analysis, we propose to test the hypothesis that functional interplays between nucleosome positioning, transcription-induced histone modifications, and SR-DNA and SR-RNA interactions may jointly define exon identity in mammalian genomes, which may also underlie the active contribution of SR proteins to transcriptional elongation. (3) We plan to use genetic approaches to determine the biological relevance of regulated splicing on animal models. We have constructed conditional knockout mice for a number of critical splicing factors and regulators. Our previous studies on SR proteins have established developing heart as a model to study regulated splicing. We recently observed a series of striking phenotype on our current knockout models, indicative of the functional importance of regulated splicing in the heart. We propose to take both genetics and genomics approaches to test the hypothesis that the splicing regulators under investigation control a splicing network critical for postnatal heart remodeling. The proposed research along this line has clear disease relevance. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed project addresses the function and regulation of SR proteins and other tissue-specific RNA binding splicing regulators in the regulation of alternative splicing on cell and animal models. The proposed research is designed to understand how exons are recognized in mammalian genomes. We propose to pursue several critical splicing regulators to define their functional requirement in establishing the splicing network during heart remodeling, which has the potential to reveal molecular pathways underlying heart diseases and other development defects in humans.